The attachment of sugar residues is one of the major modifications which a protein may undergo after synthesis of its peptide chains. A large number of proteins of diverse origin and biological function are known to contain such covalently-linked carbohydrate and are designated as glycoproteins. The role which the carbohydration steps may play in regulating protein synthesis and export and the function which the saccharide units may have in mediating fundamental cellular phenomena such as protein-membrane interactions have attracted increasing attention. Furthermore, glycoproteins have been implicated in numerous pathological states ranging from neoplasia to diabetic microangiopathy. This renewal application is being submitted to permit continuation of study designed to elucidate such fundamental aspects of glycoprotein biosynthesis as the enzymatic machinery, cellular and subcellular localization, the possibility of lipid intermediates, turnover rates, and regulatory factors. This work will continue to rely substantially on the detailed structural information obtained in this laboratory. Specific studies to be undertaken will include the purification, characterization and localization of the several mannosyl-and N- acetylglucosaminyltransferases involved in the assembly of the two types of asparagine-linked carbohydrate units of thyroglobulin and in the synthesis of the glycosylamine type of glycopeptide bond. In vivo turnover studies will be performed on the glycoprotein subunits of the renal glomerular basement membrane as well as on cellular membranes of the thyroid. The glycosyltransferase of the thyroid and kidney cortex, which have served as model systems in our previous investigations, will be employed to evaluate the physiological role of lipid intermediates and to assess the hormonal factors regulating glycoprotein biosynthesis. The study of the regulation of the basement membrane synthesizing capacity of the kidney will be a continuation of the intensive effort of this laboratory to elucidate the biochemical basis of diabetic microangiopathy.